Method for producing a virus from an african green monkey kidney cell line

ABSTRACT

A method for producing novel African Green Monkey Kidney (AGMK) cell lines is taught. These cell lines which are free of viable adventitious microbial agents are useful as substrates for viruses and for the preparation of viral vaccines.

STATEMENT AS TO RIGHTS TO THE INVENTION MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Part of the work performed during development of this invention utilizedU.S. Government funds. The U.S. Government has certain rights in thisinvention (National Institute of Allergy and Infections DiseasesContract No. N01-AO-35154).

This application is a divisional of application Ser. No. 08/790,598,filed Jan. 29, 1997, which is a division of application Ser. No.08/351,079, filed on Nov. 30, 1994, now U.S. Pat. No. 5,646,033. Thedisclosure of these applications is incorporated by reference in itsentirety herein.

FIELD OF INVENTION

The invention relates to a novel African Green Monkey Kidney (AGMK) cellsubtrate that supports the efficient replication of human and animalrotaviruses, astroviruses, enteroviruses including the Sabin liveattenuated poliovirus vaccine strains, hepatitis A virus and respiratoryviruses such as respiratory syncytial virus, parainfluenza viruses, andinfluenza A and B viruses.

The invention is also useful for the production of virus suspensionssuitable for use in vaccines for immunization of humans.

BACKGROUND OF INVENTION

Rotaviruses are major causal agents of acute gastroenteritis in man witha worldwide distribution. Enteroviruses have been implicated ininfections of the gastrointestinal tract, the respiratory system and inaseptic meningitis. Respiratory syncytial virus (RSV), the parainfluenzaviruses and the influenza viruses have been shown to be the major causalagents of serious pediatric diseases such as croup, bronchiolitis andpneumonia, while the influenza viruses are the major cause of seriousfebrile respiratory tract illness in adults. Hepatitis A virus is thecause of hepatitis A, one of the major infectious diseases of the liver.

Rotavirus infections occur world-wide and are responsible for a largeproportion of severe, life-threatening, often fatal diarrheal disease.Most rotavirus disease is caused by the four major human rotavirusserotypes but other serotypes are being discovered continuously. Thelatter are usually restricted in geographic distribution, but they couldbecome a larger problem at any time. World-wide, the human rotavirusesare the major etiologic agents of serious acute gastroenteritis ininfants and young children and on occasion can cause debilitatingdiarrhea in adults. It has been estimated by the World HealthOrganization that rotaviruses are responsible for one million fataldiarrheal illnesses each year in infants and young children indeveloping countries.

The most important cause of serious viral respiratory illness inchildren is respiratory syncytial virus (RSV). The parainfluenzaviruses, influenza viruses, and adenoviruses are also important in thisregard. Especially in the case of respiratory syncytial virus (RSV), ofwhich there are two major subgroups A and B, immunity does not appear tobe long-lasting and re-infections can and do occur with high frequencyduring infancy, through the pre-school period, and throughout adultlife. The parainfluenza viruses, of which there are four major types--1,2, 3 and 4--have been implicated as important causes of croup,bronchiolitis and pneumonia in infants and young children. These virusesare second only to RSV as a cause of severe viral respiratory tractdisease. In adults, the influenza viruses are a major cause of mortalityin older persons with underlying acute or chronic cardiac or pulmonarydisease. The serious systemic disease manifestations of influenza virusinfections are well known and the frequent antigenic shifts in serotype[A/JH1N1, A/H3N2 and B] necessitate changes in the composition of thestrains included in the yearly vaccine preparations.

The enteroviruses, which are a subgroup of picornaviruses consisting ofpolioviruses, Coxsackie viruses and echoviruses, have been shown tocause a broad spectrum of illnesses. These illnesses include paralyticdisease, encephalitis, aseptic meningitis, pleurodynia, exanthems, andpericarditis with some of the infections resulting in debilitatingsequelae. Hepatitis A virus, also a picornavirus, is a major cause ofsporadic as well as epidemic hepatitis.

According to convention, semi-continuous cell systems which are diploidand have a finite longevity in terms of the number of passages that canbe achieved in the laboratory are designated as cell strains, whereascontinuous cell systems that are aneuploid and can be passagedindefinitely in the laboratory (i.e., are imnortal) are designated ascell lines. A limiting factor in the development of vaccines forcombating the maladies caused by a number of these infectious agents hasbeen the lack of availability of an acceptable cell strain or linecapable of supporting the growth of these viruses to a concentrationsuitable for use in vaccine production. Convenient and susceptible cellstrains and/or lines that can be cultured using relatively high splitratios (i.e., the preparation of multiple tissue culture vessels from asingle culture) and which can be approved for human use by the Centerfor Biologics Evaluation and Research (CBER) of the Food and DrugAdministration (FDA) are needed to enable and facilitate vaccinedevelopment for certain medically important viruses that areuncontrolled currently.

Semi-continuous or continuous cell culture systems capable of supportingthe growth of hybrid rotaviruses, hereafter designated human x animalrotavirus reassortants, have included the simian cell strain, FRhL-2,and the simian cell lines, CV-1 and Vero. Live virus vaccines producedin these cells have received CBER, FDA approval for Phase I and IIstudies. Henceforth, we will refer to these cell strains or lines ascertified cell strains or lines to distinguish them from cell strains orlines that have been licensed for production of human vaccines. To date,only two semicontinuous cell culture systems, WI-38 and MRC-5, bothhuman fetal diploid cell strains, have been licensed for use in virusvaccines. These cell strains will be referred to as licensed cellstrains. Currently, there are no licensed continuous cell lines.

The above mentioned certified cell strain or lines (FRhL-2, CV-1 andVero) are limited in their ability to support the growth of completelyhomologous human rotaviruses, i.e., rotaviruses that derive each oftheir 11 RNA gene segments from a human rotavirus. In addition, theFRhL-2 cell strain, with a maximum 1:3 split ratio capability, islimited in its ability to support the growth of important RSV. The CV-1cells, with a maximum 1:4 split ratio capability, have not supported thegrowth of many viral agents to a level satisfactory for vaccineproduction. The Vero cells, with a split ratio of 1:6-1:10, are limitedin their ability to support the growth of a number of enteroviruses andfail to support the efficient growth of completely homologous humanrotaviruses.

Naturally occurring strains of hepatitis A virus (HAV), a picornavirusdistantly related to the enteroviruses, do not grow well in any celltype during primary isolation and must be adapted to growth in cells ofprimate origin before the level of virus replication required forvaccine development and production can be achieved. Inactivatedwhole-virus HAV vaccines grown in fetal human fibroblast MRC-5 cellshave been developed, but the low split ratio of 1:2 required by thesecells, the low viral titers achieved, and the prolonged cultivation timeof up to two weeks necessary for maximum yield of viral antigen makesuch vaccines expensive. Growth of HAV in simian CV-1, FRhK-4 (a fetalrhesus monkey kidney cell line), FRhK-6 (another fetal rhesus monkeykidney cell line) and Vero cells has been variable depending on thestrain and passage level of virus, but generally is suboptimal. Bestgrowth has been obtained in a cloned cell line, designated clone 11-1,derived from FRhK-4 cells, but these cells contain bovine papillomavirus sequences and thus, are not suitable for vaccine development.

Candidate live attenuated and inactivated HAV vaccines have beendeveloped by adaptation of the virus to growth in primary African greenmonkey (AGMK) cells but such vaccines are not economically feasiblebecause of the extreme difficulty of obtaining sufficient primary AGMKcells that are free of extraneous viral agents of monkey origin. Liveattenuated and inactivated HAV vaccine candidates have been adapted togrowth in MRC-5 cells, a human fetal diploid cell strain, but suchadaptation has consistently led, respectively, to over-attenuation ofthe live attenuated virus for humans and a relatively sparse yield ofinactivated virus vaccine.

Thus, there remains a need for a cell strain or line capable ofsupporting the efficient growth of a large number of human viralpathogens such as the human rotaviruses, enteroviruses, HAV, andrespiratory viruses of major medical importance including RSV, influenzaviruses and parainfluenza viruses. Such a cell strain or line wouldfacilitate the development and production of commercially useful andeffective vaccines. There is a pressing need for the development ofvaccines against these viruses in order to prevent severe viral diseasesof infants, children, adults and the elderly.

Therefore, the availability of a single cell strain or line capable ofsupporting the efficient replication of those viral agents responsiblefor a significant number of childhood and adult diseases would provide asignificant advancement in the formulation and production of multivalentvaccines.

SUMMARY OF THE INVENTION

The present invention is directed to a cell substrate that substantiallyovercomes the limitations of previously established cell strains orlines. The serially passaged cells of the present invention are derivedfrom the paired kidneys of an African green monkey and support theefficient growth of medically important human rotaviruses, astroviruses,picornaviruses such as enteroviruses including the Sabin live attenuatedpoliovirus vaccine strains and hepatitis A virus, and respiratoryviruses such as respiratory syncytial virus, parainfluenza viruses, andinfluenza A and B viruses.

The AGMK cells of the invention are designated as a cell substratebecause it is not clear at this time whether they constitute a cellstrain or cell line. It is probable that they are a cell line becausethey are aneuploid, an essential and defining property of cell lines.However, the AGMK cells have been passaged serially only 30 times andassignment of cell immortality, one of the other defining features of acell line, requires more than 50 serial passages.

At the 30th passage there was no diminution of viability, or ability togrow nor was there any other outward sign of senescence. Furthermore,the AGMK cells have been split 1:4 or 1:8 at each of these passages, asplit ratio that is characteristic of cell lines but not cell strainswhich usually can not be split more than 1:2.

An important advantage of the present invention is that the cellsubstrate, based on currently promulgated guidelines (i.e., "Points toConsider in the Characterization of Cell Lines Used to ProduceBiologicals" (May 1993)) and state-of-the-art testing, has beendemonstrated to be free of detectable adventitious microbial agents.This feature allows the cell substrate to be employed for the growth ofviruses, such as the human rotaviruses and hepatitis A virus, which havea very highly restricted and narrow tissue culture host range. Theunavailability of such a clean cell system prior to this invention hashampered vaccine development for these viruses. In addition, the cellsubstrate can be used in studies which require cultures of monkey kidneycells free of simian adventitious agents. The commercially availablemonkey kidney cell cultures are frequently contaminated with theseagents, which negate or becloud any results obtained and preclude theiruse in vaccines destined for administration to humans. This inventionalso offers the advantage of a cell substrate which can be trypsinizedand split in ratios ranging from 1:4 to 1:8 depending upon vessel sizeand time constraints imposed by vaccine production deadlines.

Additional features and advantages of the invention will be set forth inthe description which follows or may be learned by practice of theinvention. The objectives and other advantages of the invention to berealized and attained will be cited in the written description andclaims hereof as well as the appended drawings.

To achieve these and other advantages and in accordance with the purposeof the invention, as embodied and broadly described, the inventionprovides a cell line derived from the paired kidneys of an African greenmonkey, which is free of demonstrable adventitious microbial agents. Theinvention also provides a method for establishing the cell line by theenzymatic disaggregation of the paired kidneys from an African greenmonkey and continuous cultivation of the resulting cells. In addition,the invention provides a method for recovering and serially propagatingspecific viruses in the cell line that subsequently can be used forproduction of a viral vaccine.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 outlines the passage history from a single ampoule of adisassociated African Green Monkey Kidney sample to the preparation of aMaster Seed Cell Bank (MSCB) and a Master Working Cell Bank (MWCB).

FIG. 2 outlines the passage history from single ampoule from the MWCB topost-production history.

FIG. 3 depicts the results of a radioimmunofocus assay of HAV/7 as afraction of time for plaque formation for 11-1 cell (FRhK-derived cellline) and for AGMK cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a cell line derived from a pair ofAfrican green monkey kidneys. The cell line is free of demonstrableviable adventitious microbial agents. In addition, the cell line iscapable of supporting the growth of viruses such as human rotaviruses,astroviruses, enteroviruses, respiratory viruses and hepatitis A and is,therefore, useful for propagating viruses necessary for the productionand formulation of a number of effective vaccines on a scale that makessuch vaccines commercially feasible.

Specifically, the present invention relates to an aneuploid cellsubstrate with chromosome counts in the diploid range which wasinitiated by the enzymatic disaggregation of paired kidneys from afemale Cercopithecus African green monkey. A deposit of the cell line atpassage No. 13, Lot 6500 has been made with the American Type CultureCollection, 10801 University Boulevard, Manasses, Va. 20110-2209 USA, onNov. 4th 1994 and has been assigned accession No. ATCC# CRL 11756.

This cell line was deposited according to the Budapest Treaty. If thedeposited culture should die or otherwise become lost or destroyed, thiscell line will be replaced with a living culture containing this cellline. This cell line will be maintained for a period of at least 30years after the date of deposit, and for a period of at least 5 yearsafter the most recent request for a sample. This cell line will be madeavailable if a Patent Office signatory to the Budapest Treaty certifiesone's right to receive, or if a U.S. Patent is issued to thisapplication or any other applications claiming benefit of priority tothis application.

The present invention also provides a method of establishing a cellsubstrate capable of at least 30 serial passages that is initiallyderived by the enzymatic disaggregation of kidneys from an African greenmonkey. In addition, the invention provides a method for recovery andgrowth of specific viruses, a necessary first step for subsequentvaccine production.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the cell substrate of thepresent invention and in its uses without departing from the spirit orscope of the invention. Thus, it is intended that the present inventioncovers the modifications and variations of this invention provided theycome within the scope of the appended claims and their equivalents.

Having now described the invention in general terms, it will be betterunderstood by reference to certain examples which are included hereinfor the purpose of illustration only and are not intended to be limitingunless otherwise specified.

EXAMPLE 1 Derivation of the AGMK Cell Substrate

Source: The cell substrate was derived from the kidneys of an Africangreen monkey from a colony that had been established on Barbados. Thecells are free of any demonstrable viable adventitious microbial agents.The microbial agents include, but are not limited to bacteria, fingi,mycobacteria, mycoplasma and simian agents such as retroviruses, foamyviruses, hemadsorbing viruses, herpes-like viruses and multinucleatingor syncytial viruses. The kidneys were processed using conventionalenzymatic disaggregation technology. The resulting kidney cellsuspension was used to initiate 3 primary flask [T150 cm² ] cultureswith the remainder, designated as Freeze #2129, distributed intomultiple ampules and frozen for storage in a liquid nitrogen freezer.The primary flask cultures labelled A, B and C, were processed asfollows:

Flask A: On day 14 split 1:2=1-A-1 & 1-A-2 with one of these flaskssplit on day 28 1:2=2-A-1 & 2-A-2

Flask B: On day 21 split 1:2=1-B-1 & 1-B-2 with one of these flaskssplit on day 35 1:2=2-B-1 & 2-B-2

Flask C: Not split

The maintenance medium consisted of Eagle's Minimal Essential Medium(EMEM)+5% fetal bovine serum (heat inactivated at 56° C. for 30 min.)+1%glutamine (200 mM). All flask cultures were refed with 100 ml of freshmaintenance medium on a weekly basis for a total incubation period of 9weeks based on the initial planting date. The cultures were negative forhemadsorption and CPE (cytopathic effect), the latter determined bymicroscopic examination after fixation and staining.

The passage history from a single ampule to the preparation of theMaster Seed Cell Bank (MSCB) and the Master Working Cell Bank (MWCB) isdepicted in FIG. 1. Cultures were handled in a Class 100 Laminar FlowHood and in an area where only normal tissue cultures and/orvaccine-designated cell systems were cultured. Cultures were seriallypassaged employing a 1:4 to 1:6 split ratio with incubation at 36° C.±1° C. and utilizing the following methodology:

1. Cell films were washed with Hanks' Balanced Salt Solution (HBSS)without Ca⁺⁺ and Mg⁺⁺.

2. Films were disaggregated by the addition of a trypsin-EDTA solutionand incubation at 36° C. ±1° C. for 4-5 minutes.

3. Films were broken up by rapid pipetting with the cells suspended inthe growth medium [EMEM+10% fetal bovine serum +1% glutamine] and seededinto appropriate size culture vessels.

The passage history from single ampules from the MWCB to thepost-production testing is outlined in FIG. 2.

EXAMPLE 2 Testing of the Master Working Cell Bank - Passage 7A. Lot#4214

1. KARYOTYPING AND SPECIES IDENTIFICATION:

Passage 7A, Lot #4214 was characterized by Giemsa banded chromosomeanalysis and reaction with species-specific antisera and isoenzymeanalysis.

a. Cytogenic Characterization: Conventional chromosome staining(non-banded) was utilized to determine chromosome count ploidydistribution per 100 metaphases. Identifiable markers and aberrationswere analyzed for 50 metaphases.

Chromosome banding by trypsin-Giemsa was utilized to analyze thekaryotype and identify specific chromosome markers. Species confirmationwas made using G-banding patterns and chromosome morphology. Theprocedures used were modifications of those described by Peterson, W.D., Jr., Simpson, W. F., and Hukku, B., Cell culture characteristics:Monitoring for cell identification, in Jakoby, W. B. and Pastan, I. H.,eds., Methods in Enzymology 58: 164-178, 1979; and Seabright, M., Arapid banding technique for human chromosomes, Lancet, ii: 971-972,1971.

The cell line was identified as an aneuploid female Cercopithecus(African green) monkey (XX) with chromosome counts in the diploid range.Most of the chromosomes were normal and present in two copies each.Normal chromosome D was monosomic and a single marker D chromosome wasobserved.

b. Species Identification of Cell Culture: The ability of the test cellpreparation to react with species-specific antisera was determined usingthe fluorescent antibody technique as described by Peterson et al.,supra. The cells reacted with monkey antiserum but not with mouse orhamster antiserum.

C. Isoenzyme Analysis: Enzyme preparations were made from the testarticle cells. The electrophoretic migration distances of the enzymeswere compared with known migration distances of enzymes in variousmammalian species as described by Halton, D. M., Peterson, W. D., Jr.,and Hukku, B. Cell culture quality control by rapid isoenzymaticcharacterization, In Vitro 19: 16-24, 1983; Ottenbreit, M. J., Halton,D. M., and Peterson, W. D., Jr., Rapid isoenzyme analysis of cellcultures by agarose electrophoresis, I. Interspecies identification, JTissue Culture Methods, 6: 107-110, 1982; and II. Intraspeciesidentification of human cell lines, J. Tissue Culture Methods, 8:125-130, 1986.

The electrophoretic mobilities of enzymes glucose-6-phosphatedehydrogenase (G6PD), nucleoside phosphorylase (NP), malatedehydrogenase (MDH) and lactate dehydrogenase (LDH) present in anextract prepared from the test cells were comparable to those of aCercopithecus (African green) monkey control cell preparation. No extrabands were observed on the electrophoresis films that might suggest thepresence of cells of a species other than that of the cell culture.

Cells other than those of the submitted culture (MWCB, Passage 7A,Lot#4214) were not detected in the culture by either isoenzyme analysisor by cytogenic evaluation.

2. DETECTION OF RETROVIRUSES

a. Detection of Retrovirus Particles by Electron MicroscopicExamination: A cell pellet of the test article cells (MWCB, Passage 7A,Lot#4214) was prepared and examined for the presence of virus particlesby transmission electron microscopy. No retrovirus-like particles werefound in the 200 cells observed.

b. Detection of Retrovirus Reverse Transcriptase in the Presence ofDNA-Dependent DNA Polymerase: The test article (MWCB, Passage 7A,Lot#4214) was analyzed for the presence of types B, C and D retrovirusreverse transcriptase and cellular DNA polymerase activity. No evidencefor the presence of retrovirus reverse transcriptase was observed.

The results are presented in Table I--A, B and C.

                                      TABLE I-A                                   __________________________________________________________________________    Reverse Transcriptase and DNA Polymerase Activities                            in Test Article AGMK MWCB Cell Cultures: p7A, L-4214                                       Adjusted Reverse                                                                       Reverse  DNA                                             Transcriptase.sup.a Transcriptase.sup.b Polymerase.sup.b                      (rAdT) (rAdT) (dAdT)                                                        Sample        Mn.sup.++                                                                              Mn.sup.++                                                                         Mn.sup.++                                                                          Mn.sup.++                                                                        Mn.sup.++                                  __________________________________________________________________________    Test Article                                                                    Undiluted 22 25 0 7 0                                                         Diluted 2-fold in medium.sup.c 6 8 0 19 1                                     Diluted 2-fold in R-MuLV.sup.d  20,837 433 10 0                               Diluted 2-fold in  795 6 7,137 508                                            DNA polymerase.sup.e                                                          Positive and Negative Controls.sup.f                                          R-MuLV diluted  18,589 549 12 0                                               2-fold in medium.sup.g                                                        DNA Polymerase diluted  1,079 25 5,443 542                                    2-fold in medium.sup.g                                                        SMRV.sup.h  2,585 48,289 211 396                                              Stabilization Buffer.sup.i  (63) (59) (67) (58)                               Medium.sup.c  (65) (57) (65) (52)                                             Medium.sup.g  (78) (56) (62) (49)                                           __________________________________________________________________________     .sup.a Reverse transcriptase activity adjusted for the effect of DNA          polyrnerase (Table 2 and Methods).                                            .sup.b Expressed as counts per minute .sup.3 Hthymidine triphosphate          incorporated (mean duplicates minus background).                              .sup.c EMEM + 10% serum subtracted from test article samples.                 .sup.d RMuLV (Rauscher Murine Leukemia Virus)  Type C virus positive          control.                                                                      .sup.e DNA polymerase  Positive control; prepared from mink lung cells.       .sup.f Control monitoring background of incorporation of .sup.3 Hthymidin     triphosphate (reaction mixture with undiluted test article minus template     primers) = 61 cpm (medium background not subtracted).                         .sup.g EMEM + 10% serum subtracted from RMeLV and DNA polymerase positive     controls.                                                                     .sup.h SMRV (Squirrel Monkey Retrovirus Preparation)  Type D virus            positive control.                                                             .sup.i Stabilization buffer used to dilute SMRV positive control.        

                  TABLE I-B                                                       ______________________________________                                        Adjustment of Reverse Transcriptase Values for Interference by                  Polymerase in Test Article AGMK MWCB Cell Cultures: p7A, L-4214                                DNA Polymerase Assay (CPM)                                        RT Assay (CPM)        DNA                                                     Observed.sup.a                                                                       Adjusted.sup.b                                                                         Observed  Polymerase ×                             (Mn.sup.++) (Mn.sup.++) (Mn.sup.++) Slope                                   ______________________________________                                        Test Article                                                                    Undiluted 25 22 37 3                                                          Diluted 2-fold 8 6 19 2                                                       in medium.sup.c                                                             ______________________________________                                         .sup.a Unadjusted .sup.3 HTTP incorporation catalyzed by RT. May include      effects by cellular DNA polymerase.                                           .sup.b Adjusted .sup.3 HTTP incorporation catalyzed by RT = Observed RT       (DNA polymerase × Slope). Slope = 0.08.                                 .sup.c Medium used to dilute test article.                               

                  TABLE I-C                                                       ______________________________________                                        Reverse Transcriptase and DNA Polymerase Activities                             in a DNA Polymerase Preparation from Reverse                                  Transcriptase-Negative Cell Cultures                                          (Used for Determining Interference in Reverse                                 Transcriptase Assay by DNA Polymerase)                                                    Reverse Transcriptase.sup.b                                                                DNA Polymerase.sup.b                                 (rAdT) (dAdT)                                                               DNA Polymerase.sup.a                                                                        Mn.sup.++                                                                            Mg.sup.++ Mn.sup.++                                                                           Mg.sup.++                                ______________________________________                                        Sample 1      116    10        1,898 198                                        Sample 2 187 0 2,634 0                                                        Sample 3 547 6 6,690 119                                                      Sample 4 574 3 8,529 81                                                       Sample 5 1,196 27 14,315 1,222                                              ______________________________________                                         .sup.a DNA polymerase prepared from mink lung cells.                          .sup.b Expressed as counts per minute .sup.3 Hthymidine triphosphate          incorporated [mean duplicates minus background (medium)].                

EXAMPLE 3 Post-Production Cell Testing

1. Karyotyping and Species Identification

The AGMK cells at Passage 17A, Lot #4706 were characterized by Giemsabanded chromosome analysis and reaction with species-specific antiseraand isoenzyme analysis.

The cells were identified as aneuploid female Cercopithecus (Africangreen) monkey (XX, XXX) with chromosome counts in the hypodiploid tohyperdiploid range. All karyotypes examined contained a single normalCercopithecus monkey marker chromosome (Group D) and a second partiallydeleted Cercopithecus monkey D group marker chromosome.

The electrophoretic mobilities of the enzymes G6PD, NP, MDH and LDHpresent in an extract prepared from the test cells were comparable tothose of Cercopithecus (African green) monkey control cell preparation.No extra bands were observed on the electrophoresis films that mightsuggest the presence of cells of a different species in the cellculture. The cells reacted with monkey antiserum but not with mouseantiserum.

Cells other than those of the submitted culture (Passage 17A, Lot #4706)were not detected in the culture by either antisera reaction, isoenzymeanalysis or by cytogenic evaluation. The chromosome pattern was similarto that previously observed for cells in the MWCB, Passage 7A, Lot #4214and specifically related these cells (Passage 17A, Lot #4706) to theformer.

2. Detection of Retroviruses

a. The test article, AGMK Passage 17A, Lot #4706, was analyzed for thepresence of types B, C and D retrovirus reverse transcriptase andcellular DNA polymerase activity. The cells were grown in the presenceof 30 μg/ml bromodeoxyuridine for 24 hours (day 0 to day 1) and thenrefed with medium without inducer. Culture fluids (unconcentrated andconcentrated 20-fold) were harvested on days 2, 3 and 4. No evidence forthe presence of retrovirus reverse transcriptase activity was observedin the test article. The results are presented in Table II--A, B and C.

b. Detection of Retrovirus Particles by Electron MicroscopicExamination: Cell pellets were prepared from the treated and untreatedcultures harvested on day 4 and examined for the presence of virusparticles by transmission electron microscopy. No retrovirus-likeparticles were found in the 200 cells observed.

c. Detection of Simian T-Lymphotropic Virus (STLV) in Test Article(AGMK, Passage 17A, Lot #4743) by Polymerase Chain Reaction (PCR)Technique: The polymerase chain reaction (PCR) provides a method fordetection of extremely low numbers of viral nucleic acid sequences,which would not be detectable without amplification. When coupled with apre-PCR reverse transcriptase step, RNA sequences may be amplified, thusallowing detection of free virus. The protocol used allowed for theamplification of either DNA or RNA derived STLV-specific sequences. ThePCR products were tested by Southern blotting, using chemiluminescentenzyme-linked probe hybridization to the blot.

When tested with the STLV pot primers SK 110/SK111, STLV-specificproduct was not detected in test article reactions, each of whichrepresented 1.0 μg nucleic acid extracted from the test article. In thepositive control series, which contained the equivalent of 0.001 μg,0.01 μg, 0.1 μg or 1 μg of nucleic acid extracted from a cell lineinfected by STLV (STLV_(mn103)), STLV-specific product was detected inall reactions. On the basis of failure to detect reactivity with STLVprimers, the test article was judged to be negative for STLV nucleicacid sequences.

d. Detection of the Simian Immunodeficiency Virus in Test Article (AGMK,Passage 18A, Lot #5506): Total cellular DNA was extracted from the testarticle and subsequently tested for the presence of SIV_(agm) virussequences using primers that amplify a 200 bp fragment of the LTR. Thisinvolved PCR with nested sets of primers as follows:

    Outer set                                                                           #1807 F: CCTCAGAGCTGCATAAAAGCAGAT (SEQ ID NO:1)                            -  #1808 R: TCACTCAAGTCCCTGTTCGGGCGC (SEQ ID NO:2)                            - Inner set #1809 F: TACTAGGAGACCAGCTTGAGCCTG (SEQ ID NO:3)                   -  #1810 R: TGCTGGAGTTTCTCTCGCCTGGGT (SEQ ID NO:4)                     

These primers were designed for the conserved U5 and R regions of theLTR and are capable of amplifying all subtypes of SIV_(agm). Thepredicted 200 bp fragment was amplified from DNA extracted from achronically-SIV_(agm) infected cell line (SIV_(agm) 155CEM_(ss)) but theAfrican green monkey cells did not have detectable SIV_(agm) sequencesby Southern blot analysis.

3. Tumorigenicity in Athymic Nude Mice

The cells were found to be non-tumorigenic after being clinicallyevaluated for 150 days. Athymic nude mice were inoculated subcutaneouslywith approximately 1×10⁷ test article cells (AGMK Passage 17A, Lot#4889). Athymic nude mice were similarly inoculated with positivecontrol cells. All 10 positive control inoculated mice had tumors at thesite of inoculation. No metastases were observed in the inoculation site(skin), lung, lymph nodes, liver, kidney, spleen or brain of miceinoculated with the test article.

                  TABLE II-A                                                      ______________________________________                                        Reverse Transcriptase and DNA Polymerase Activities                             in Unconcentrated AGMK Passage 17A, Lot #4706                                              Reverse Transcriptase.sup.b                                                                DNA Polymerase.sup.b                                (rAdT) (dAdT)                                                               Sample         Mn.sup.++                                                                            Mg.sup.++ Mn.sup.++                                                                          Mg.sup.++                                ______________________________________                                        Test Article Cells.sup.b                                                          DAY                                                                         2 Untreated 0 0 227 0                                                          Treated 29 0 485 85                                                          3 Untreated 0 0 265 6                                                          Treated 0 0 243 0                                                            4 Untreated 35 0 711 29                                                        Treated 48 0 723 26                                                        ______________________________________                                         .sup.a Expressed as counts per minute .sup.3 Hthymidine triphosphate          incorporated (mean duplicates minus background).                              .sup.b Culture fluids from test article cells.                           

                  TABLE II-B                                                      ______________________________________                                        Reverse Transcriptase and DNA Polymerase Activities                             in AGMK Passage 17A, Lot #4706                                                Concentrated 20-fold                                                                       Reverse Transcriptase.sup.b                                                                DNA Polymerase.sup.b                                (rAdT) (dAdT)                                                               Sample         Mn.sup.++                                                                            Mg.sup.++ Mn.sup.++                                                                          Mg.sup.++                                ______________________________________                                        Test Article Cells.sup.b                                                          DAY                                                                         2 Untreated 9 0 18 87                                                          Treated 0 0 15 104                                                           3 Untreated 6 0 32 125                                                         Treated 9 3 19 136                                                           4 Untreated 22 3 14 114                                                        Treated 122 2 2,423 516                                                    ______________________________________                                         .sup.a Expressed as counts per minute .sup.3 Hthymidine triphosphate          incorporated (mean duplicates minus background).                              .sup.b Culture fluids from test article cells.                           

                  TABLE II-C                                                      ______________________________________                                        Reverse Transcriptase and DNA Polymerase Activities                             in AGMK Passage 17A, Lot #4706                                                             Reverse Transcriptase.sup.a                                                                DNA Polymerase.sup.b                                (rAdT) (dAdT)                                                               Sample         Mn.sup.++                                                                              Mg.sup.++                                                                             Mn.sup.++                                                                            Mg.sup.++                              ______________________________________                                        Positive and Negative                                                           Controls                                                                      R-MuLV.sup.c 65,913 2,230 0 16                                                SMRV.sup.d 12,031 133,798 1,274 2,825                                         DNA Polymerase.sup.e 7,015 57 60,197 6,646                                    Medium.sup.f (163) (74) (242) (107)                                           Stabilization Buffer.sup.g (53) (59) (46) (44)                                Medium.sup.h (146) (136) (321) (148)                                        ______________________________________                                         .sup.a Expressed as counts per minute .sup.3 Hthymidine triphosphate          incorporated (mean duplicates minus background).                              .sup.b Culture fluids from test article cells.                                .sup.c RMuLV (Rauscher murine leukemia virus)  Type C virus positive          control.                                                                      .sup.d SMRV (Squirrel Monkey Retrovirus Preparation)  Type D virus            positive control.                                                             .sup.e DNA polymerase  Positive control; prepared from mink lung cells.       .sup.f Medium  Subtracted from unconcentrated, untreated and treated          samples.                                                                      .sup.g Stabilization buffer  Negative control; subtracted from positive       controls and concentrated samples.                                            .sup.h Medium  Subtracted from RMulV and DNA polymerase positive controls

EXAMPLE 4 Post-Production Testing for Microbial Sterility

1. Bacterial Sterility in Fluid Thioglycollate Medium

Each of 10 culture tubes (9-10 ml medium per tube) w as inoculated with1.0 ml of the AGMK cell suspension (Passage 17A, Lot #4706) containingapprox. 5×10⁵ cells per ml. Each of 5 tubes was inoculated with 1.0 mlof the original growth medium. Ten cultures were included asuninoculated controls. All cultures were vortex mixed and incubated at32° C. ±2° C. for 21 days with observations made on days 1, 3, 6, 8, 10,13, 15, 17, 20 and 21. No growth was observed in any of the 25 cultures.

2. Fungal Sterility in Soybean Casein Digest Medium

Each of 10 culture tubes (9-10 ml medium per tube) was inoculated with1.0 ml of the AGMK cell suspension (Passage 17A, Lot #4706) containingapprox. 5×10⁵ cells per ml. Each of 5 tubes was inoculated with 1.0 mlof the original growth medium. Ten cultures were included asuninoculated controls. All cultures were vortex mixed and incubated at22° C.±2° C. for 21 days with observations made on days 1, 3, 6, 8, 10,13, 15, 17, 20 and 21. No growth was observed in any of the 25 cultures.

3. Mycobacterial Sterility in Lowenstein-Jensen Egg Medium

Each of 10 slant culture tubes was inoculated with 0.5 ml of the AGMKcell suspension (Passage 17A, Lot #4706) containing approx. 5×10⁵ cellsper ml. Each of 10 culture slants was inoculated with 0.5 ml of theoriginal growth medium. Six cultures were included as uninoculatedcontrols. All cultures were incubated at 36° C.±1° C. horizontally forthe first 24 hours and vertically for the remainder of the 8 weekobservation period with weekly observations for growth. On day 28, onecell suspension inoculated culture was found contaminated with a mold.No growth was observed in any of the remaining 25 cultures after 8weeks.

This assay was repeated with AGMK Passage 18A, Lot #4918, but using 6culture slants inoculated with the original growth medium. No growth wasobserved in any of the 22 cultures after 8 weeks. The results of theabove Microbial Sterility Assays are summarized in Table III.

4. Mycoplasma Sterility

The assay for mycoplasma was performed using both the direct BrothEnrichment and Agar Procedures and the indirect Indicator Cell CultureProcedure with the AGMK cell suspension (Passage 17A, Lot #4706)containing approximately 5×10⁵ cells per ml. The cell suspension wasfound to be negative for mycoplasmas by all procedures.

                                      TABLE III                                   __________________________________________________________________________    Microbial Sterility Test Results on the AGMK Cell Suspension -                  Passage 17A, Lot #4706 and #4918 at Approximately 5 × 10.sup.5        Cells/ml                                                                                   Vol. Per                                                           Culture Date                                                                Culture Medium                                                                             No. (ml)                                                                             Temp.                                                                             On Test                                                                           Off Test                                                                          Results                                       __________________________________________________________________________    Fluid Thioglycollate                                                            (FIM) LOT VVPL #122-50 10 -- 32° C. 1/05/93 1/26/93 No Growth                                         Cell Suspension #4706 10 1.0 (±2.degre                                    e. C.) | | No Growth                                         Culture Medium 5 1.0  1/05/93 1/26/93 No                                     Growth                                          Soybean-Casein-Digest                                                         (SCDM) LOT VVPL #112-51 10 -- 22° C. 1/05/93 1/26/93 No Growth                                        Cell Suspension #4706 10 1.0 (±2.degre                                    e. C.) | | No Growth                                         Culture Medium 5 1.0  1/05/93 1/26/93 No                                     Growth                                          Lowenstein-Jensen Egg                                                         Medium - LOT #C3CPPB 6 -- 36° C. 1/05/93 3/02/93 No Growth                                            Cell Suspension #4706 10 0.5 (±1.degre                                    e. C.) | | 1/10 mold                                         Culture Medium 10 0.5  |                                            | on day 28                                1/05/93 3/02/93 No Growth                                                 Lowenstein-Jensen Egg                                                         Medium - LOT #H3CTGX 6 -- 36° C. 2/23/93 4/20/93 No Growth                                            Cell Suspension #4918 10 0.5 (±1.degre                                    e. C.) | | No Growth                                         Culture Medium 6 0.5  2/23/93 4/20/93 No                                     Growth                                        __________________________________________________________________________

EXAMPLE 5 In Vitro Tissue Culture Purity and Safety Tests for theDetection of Viable Adventitious Microbial Agents

These assays were performed in roller tube cultures of the followingtissue culture systems: Serially passaged African green monkey kidney(AGMK); Primary Human Amnion (PHA); Vero; Primary Rabbit Kidney (PRK);Whole Human Embryo Fibroblast (Fl 5000); and Human Carcinoma of theLarynx (H.Ep-2). Cultures were maintained on Medium EMEM containing2-10% fetal bovine serum (gamma irradiated) plus antibiotics:gentamicin, 100 μg/ml; neomycin, 50 μg/ml; and fungizone, 2.5 μg/ml.Cultures were refed with 2 ml of maintenance medium prior toinoculation.

Testing Procedures: The cell suspensions (Lots #4742/3, #4755 or #4889containing approximately 5×10⁵ cells per ml) were inoculated in 0.5 mlamount per each of 28 roller tubes per tissue culture system. Cultureswere incubated at 36° C.±1° C. for 14 days with periodic microscopicexamination for any cytopathic effect (CPE) and/or cellulardegeneration. When necessary to maintain the integrity of the cellfilms, cultures were refed with 2 ml of fresh maintenance medium.Additional groups of 28 roller tubes per tissue culture system wereincluded as uninoculated controls.

On the 7th-8th day of incubation, 6-9 tubes from each series (inoculatedand controls) were removed and tested for hemadsorption with 3 red bloodcell (RBC) suspensions (guinea pig at 0.1%, chicken at 0.075%, or humanat 0.1% in PBS, pH 7.2). Films were rinsed once in thephosphate-buffered saline (PBS) prior to the addition of 1 ml of the RBCsuspension employing 2-3 tubes per RBC. Initially, tubes were incubatedat 2°-8° C. for a minimum of 30 minutes and examined microscopically forany signs of hemadsorption. Following manual shaking, tubes wereincubated at 35°-37° C. for a minimum of 30 minutes and re-examinedmicroscopically for hemadsorption. All cultures were inactive forhemadsorption with all 3 suspensions and at both temperatures. On day14, after final microscopic examination, the cultures were divided,depending on the specific tissue culture system for final testing. AGMK,PHA and F15000 Tube Cultures were divided as follows:

1. 9 tubes from each series were tested for hemadsorption with the 3RBCsuspensions as described above for day 7-8;

2. 4 tubes from each series were fixed and stained with a solution of 5%glutaraldehyde +0.025% crystal violet and, after drying, examinedmicroscopically for any signs of CPE;

3. 8 tubes from each series were challenged with Coxsackie A-9 virus(0.1 ml for each of 2 tubes using a 10⁻², 10⁻³, 10⁻⁴ or 10⁻⁵ dilution)for the detection of non-CPE producing agents and/or latent agents viathe interference phenomenon; and

4. One (1) tube of each series was included as an uninoculated controlfor the challenge virus.

Vero, PRK and H.Ep-2 Tube Cultures were divided as follows:

1. 12 tubes from each series were tested for hemadsorption with the 3RBC suspensions as described above for day 7-8;

2. 7 tubes from each series were fixed and stained with a solution of 5%glutaraldehyde +0.025% crystal violet and, after drying, examinedmicroscopically for any signs of CPE.

No challenge studies were carried out with the Coxsackie A-9 virus asthis agent does not produce any discernible CPE in these cell systems.The results of these in vitro tissue culture purity and safety assaysare summarized in Tables IV--A to F.

                                      TABLE IV-A                                  __________________________________________________________________________    Tissue Culture Purity Tests on the AGMK Cell Suspension -                       Passage 17A, LOT #4742/3 at Approximately 5 × 10.sup.5 Cells/ml        African Green Monkey Kidney Cells - (Passage 12, LOT #4793)                                          Day 7 Results                                                                          Day 14 Results                                                       AGMK                                                                              Controls                                                                           AGMK                                                                              Controls                                 __________________________________________________________________________    0.1% Guinea Pig RBC                                                                         at  2°-8° C.                                                              0/2 0/2  0/3 0/3                                         at 35°-37° C. 0/2 0/2 0/3 0/3                                  0.075% Chicken RBC at 2°-8° C. 0/2 0/2 0/3 0/3                   at 35°-37° C. 0/2 0/2 0/3 0/3                                  0.1% Human RBC at 2°-8° C. 0/2 0/2 0/3 0/3                       at 35°-37° C. 0/2 0/2 0/3 0/3                                  Giemsa Stain     0/4 0/4                                                      Coxsackie A-9 Virus Challenge* at 10.sup.-3    2/2 2/2                         10.sup.-4    2/2 2/2                                                          10.sup.-5    2/2 2/2                                                          10.sup.-6    2/2 2/2                                                                     Unchallenged Controls                                                                            0/1 0/1                                      __________________________________________________________________________     *Coxsackie A9 Challenge results based on a 5day incubation at 36°      C. Prior to challenge, tubes refed with 2 ml of fresh medium.            

                                      TABLE IV-B                                  __________________________________________________________________________    Tissue Culture Purity Tests on the AGMK Cell Suspension -                       Passage 17A, LOT #4742/3 at Approximately 5 × 10.sup.5 Cells/ml        Primary Human Amnion - (PHA) - (LOT #4720)                                                           Day 7 Results                                                                          Day 14 Results                                                       AGMK                                                                              Controls                                                                           AGMK                                                                              Controls                                 __________________________________________________________________________    0.1% Guinea Pig RBC                                                                         at  2°-8° C.                                                              0/2 0/2  0/3 0/3                                         at 35°-37° C. 0/2 0/2 0/3 0/3                                  0.075% Chicken RBC at 2°-8° C. 0/2 0/2 013 0/3                   at 35°-37° C. 0/2 0/2 0/3 0/3                                  0.1% Human RBC at 2°-8° C. 0/2 0/2 0/3 0/3                       at 35°-37° C. 0/2 0/2 0/3 0/3                                  Giemsa Stain     0/4 0/4                                                      Coxsackie A-9 Virus Challenge* at 10.sup.-3    2/2 2/2                         10.sup.-4    2/2 2/2                                                          10.sup.-5    2/2 212                                                          10.sup.-6    2/2 2/2                                                                     Unchallenged Controls                                                                            0/1 0/1                                      __________________________________________________________________________     *Coxsackie A9 Challenge results based on a 5day incubation at 36°      C. Prior to challenge, tubes refed with 2 ml of fresh medium.            

                                      TABLE IV-C                                  __________________________________________________________________________    Tissue Culture Purity Tests on the AGMK Cell Suspension -                       Passage 17A, LOT #4755 at Approximately 5 × 10.sup.5 Cells/ml          Vero Cells - (Passage 143, LOT #4813)                                                                Day 7 Results                                                                          Day 14 Results                                                       AGMK                                                                              Controls                                                                           AGMK                                                                              Controls                                 __________________________________________________________________________    00.1% Guinea Pig RBC                                                                        at  2°-8° C.                                                              0/3 0/3  0/4 0/4                                         at 35°-37° C. 0/3 0/3 0/4 0/4                                  0.075% Chicken RBC at 2°-8° C. 0/3 0/3 0/4 0/4                   at 35°-37° C. 0/3 0/3 0/4 0/4                                  0.1% Human RBC at 2°-8° C. 0/3 0/3 0/4 0/4                       at 35°-37° C. 0/3 0/3 0/4 0/4                                              Giemsa Stain       0/7 0/7                                      __________________________________________________________________________

                                      TABLE IV-D                                  __________________________________________________________________________    Tissue Culture Purity Tests on the AGMK Cell Suspension -                       Passage 17A, LOT #4889 at Approximately 5 × 10.sup.5 Cells/ml          Rabbit Kidney Cells - (Primary, LOT #4814)                                                           Day 7 Results                                                                          Day 14 Results*                                                      AGMK                                                                              Controls                                                                           AGMK                                                                              Controls                                 __________________________________________________________________________    00.1% Guinea Pig RBC                                                                        at  2°-8° C.                                                              0/4 0/4  0/3 0/3                                         at 35°-37° C. 0/4 0/4 0/3 0/3                                  0.075% Chicken RBC at 2°-8° C. 0/4 0/4 0/3 0/3                   at 35°-37° C. 0/4 0/4 0/3 0/3                                  0.1% Human RBC at 2°-8° C. 0/4 0/4 0/3 0/3                       at 35°-37° C. 0/4 0/4 0/3 0/3                                              Giemsa Stain       0/7 0/7                                      __________________________________________________________________________     *On day 8, tubes refed with 2 ml of fresh medium.                        

                                      TABLE IV-E                                  __________________________________________________________________________    Tissue Culture Purity Tests on the AGMK Cell Suspension -                       Passage 17A, LOT #4889 at Approximately 5 × 10.sup.5 Cells/ml          Whole Human Embryo Fibroblast Cells - (FL 5000) - (Passage 22, LOT           #4842)                                                                                                Day 7 Results                                                                          Day 14 Results                                                       AGMK                                                                              Controls                                                                           AGMK                                                                              Controls                                 __________________________________________________________________________    0.1% Guinea Pig RBC                                                                         at  2°-8° C.                                                              0/2 0/2  0/2 0/2                                         at 35°-37° C. 0/2 0/2 0/2 0/2                                  0.075% Chicken RBC at 2°-8° C. 0/2 0/2 0/2 0/2                   at 35°-37° C. 0/2 0/2 0/2 0/2                                  0.1% Human RBC at 2°-8° C. 0/2 0/2 0/2 0/2                       at 35°-37° C. 0/2 0/2 0/2 0/2                                                  Giemsa Stain   0/4 0/4                                      Coxsackie A-9 Virus Challenge*                                                              at 10.sup.-3       2/2 2/2                                         10.sup.-4 2/2 2/2                                                             10.sup.-5 2/2 2/2                                                             10.sup.-6 1/2 1/2                                                             Unchallenged Controls 0/1 0/1                                              __________________________________________________________________________     *Coxsackie A9 Challenge results based on a 4day incubation at 36°      C. Prior to challenge, tubes refed with 2 ml of fresh medium.            

                                      TABLE IV-F                                  __________________________________________________________________________    Tissue Culture Purity Tests on the AGMK Cell Suspension -                       Passage 17A, LOT #4755 at Approximately 5 × 10.sup.5 Cells/ml          Human Carcinoma of the Larynx - H.Ep-2 - (Passage 397, LOT #4808)                                    Day 7 Results                                                                          Day 14 Results*                                                      AGMK                                                                              Controls                                                                           AGMK                                                                              Controls                                 __________________________________________________________________________    0.1% Guinea Pig RBC                                                                         at  2°-8° C.                                                              0/3 0/3  0/4 0/4                                         at 35°-37° C. 0/3 0/3 0/4 0/4                                  0.075% Chicken RBC at 2°-8° C. 0/3 0/3 0/4 0/4                   at 35°-37° C. 0/3 0/3 0/4 0/4                                  0.1% Human RBC at 2°-8° C. 0/3 0/3 0/4 0/4                       at 35°-37° C. 0/3 0/3 0/4 0/4                                    Giemsa Stain   0/7 0/7                                                    __________________________________________________________________________     *On day 10, tubes refed with 2 ml of fresh medium.                       

EXAMPLE 6 In Vivo Tests for the Detection of Viable AdventitiousMicrobial Agents

The following tests were performed employing cell suspensions derivedfrom Passage 17A--(Lots #4706, #4742, #4755 and #4788). These assayswere performed with inocula consisting of approximately 1×10⁷ cells perml suspended in the culture medium in which they were grown.

1. Tests in Animals

a. Suckling Mouse Assay: A total of 10 newborn CD-1 mice randomized fromdifferent litters (10 per mother, less than 24 hours old) wereinoculated intracerebrally (I.Cer.) with 0.01 ml and intraperitoneally(I.P.) with 0.1 ml of the cell suspension from Lot #4706. An additionalrandomized group of 10 sucklings was inoculated in a similar manner withthe original culture medium. A randomized group of 10 sucklings wasincluded as uninoculated controls. All sucklings were observed daily for14 days for deaths and/or signs of illness or distress. On day 2, one(1) suckling from each of the inoculated groups was found missing andpresumed cannibalized. There were no other deaths and none of thesucklings exhibited any signs of illness or distress over this initial14 day observation period.

On the 14th day, single pools were prepared of the emulsified tissue(minus skin and viscera) of the following groups: (a) AGMK inoculatedpups (n=9); (b) culture fluid inoculated pups (n=9); and (c)uninoculated controls (n=10). A blind passage into newborn CD-1 mice wasmade of each pool via the I.Cer. and I.P. routes: each pool into 10newborns from mixed litters. An additional litter of 10 sucklings wasincluded as uninoculated controls for this blind passage. All sucklingswere observed daily for 14 days for deaths and/or signs of illness ordistress. There were no deaths recorded for the AGMK or culture mediuminoculated pups or for the uninoculated control group; however, on day3, 4 sucklings were missing and presumed cannibalized in (c) thepassaged control group. None of the sucklings exhibited any signs ofillness or distress over this final 14 day observation period.

Because none of the AGMK cell suspension or culture medium inoculatedsucklings exhibited any signs of a transmissible agent or of Coxsackievirus infection or of any viral infection, and because >90% of theseinoculated sucklings (19 of 20) remained healthy and survived the entireobservation period, this assay in suckling mice was consideredsatisfactory.

b. Adult Mouse Assay: Each of 20 adult CD-1 mice (VAF, 15-20 grams each)was inoculated I.Cer. and I.P. with 0.03 ml and 0.5 ml, respectively,with the cell suspension Lot #4788. An additional 5 mice were inoculatedin a similar manner with the original culture medium. Four mice wereincluded as uninoculated controls. Mice were observed daily for deathsand/or signs of illness or distress over a 21 day period. There were nodeaths recorded and all mice survived the entire 21 day observationperiod with no evidence of lymphocytic choriomeningitis virus infectionor any other viral infection. This test in adult mice was consideredsatisfactory.

c. Adult Guinea Pig Assay: Each of 5 Hartley strain guinea pigs (VAF,350-450 grams each] was inoculated I.Cer. and I.P. with 0.1 ml and 5.0ml, respectively, with the cell suspension Lot #4755. An additionalguinea pig was inoculated in a similar manner with the original culturemedium. One (1) guinea pig was included as an uninoculated control. Allwere observed daily for deaths and/or signs of illness or distress overa 6 week period; none were recorded. Commencing on day 21, daily rectaltemperatures were taken with a digital thermometer and recorded (atapproximately 0900 hours) for all guinea pigs until time of sacrifice.The average temperatures (° C.) for the AGMK inoculated guinea pigswere: 39.26, 39.30, 39.40, 39.46 and 39.53; for the medium inoculatedguinea pig: 39.32; and for the control: 39.37. There were no significanttemperature rises indicative of either bacterial or viral infection. Allguinea pigs appeared healthy and survived the entire 42-day observationperiod at which time they were necropsied following euthanasia withHalothane and exsanguination. Inspection of the abdominal and thoraciccavities by the consulting veterinarian did not identify gross lesionsor pathological changes. This assay in adult guinea pigs was consideredsatisfactory.

d. Adult Rabbit Assay: Each of 5 New Zealand white rabbits (1500-2500 gmeach) was inoculated intradermally (I.D.) with 0.1 ml per each of 10sites and subcutaneously (S.Q.) with 9.0 ml of the AGMK cell suspensionLot #4788. One (1) rabbit was similarly inoculated with the originalculture medium. One (1) rabbit was included as an uninoculated control.Animals were observed daily for 28 days for deaths, illness, distressand/or lesions at the sites of inoculation. No deaths were recorded andall rabbits remained healthy without exhibiting any signs of illness ordistress or lesions at the sites of inoculation. This test in adultrabbits was considered satisfactory. The results of these in vivo animalsafety tests are summarized in Table V--A and B.

2. Tests in Embryonated Eggs

For these studies, specific pathogen-free embryonated eggs were obtainedfrom SPAFAS, Inc., Reinholds, PA. These eggs came from Flock L061-E.

a. Allantoic Inoculation: Each of ten 10-day old embryonated eggs wasinoculated via the allantoic route with 0.5 ml of the AGMK suspensionLot #4706. An additional 5 eggs were inoculated in a similar manner withthe original culture medium. Five (5) eggs were included as uninoculatedcontrols. Eggs were incubated at 36° C.±1° C. for 72 hours and thencandled for deaths (1 of 5 medium inoculated) and chilled overnight at2°-8° C. Allantoic fluids were harvested individually, incubated in a37° C. water bath for 60 minutes to elute any adsorbed agent(s), andthen clarified by centriflugation at 2500 rpm for 15 minutes at 20° C.Pools were prepared for each of the 3 sets of eggs employing equalvolumes from each individual harvest. The pools were assayed forhemagglutination both undiluted and at a 1:10 dilution with incubationat 2°-8° C. and at room temperature (15°-+° C.) using the followingerythrocytes in PBS at (pH 7.2): guinea pig at 0.6%; chick at 0.4%; andhuman at 0.6%. All fluids were negative for hemagglutination at bothdilutions and at both temperatures with all 3 RBC suspensions.

The sample pools were subpassaged in 0.5 ml amounts into 10-day oldembryonated eggs via the allantoic route as follows: the pool from theAGMK cell suspension into 10 eggs; the pool from the medium into 5 eggs;and the pool from the uninoculated eggs into 5 eggs. An additional 5eggs were included as uninoculated controls. Eggs were incubated at 36°C.±1° C. for 72 hours and then candled for deaths (none recorded) andchilled overnight at 2°-8° C. Allantoic fluids were harvested, handledand tested as described above. All 4 pools were negative forhemagglutination at both dilutions and at both temperatures with all 3RBC suspensions.

There were no deaths recorded for any of the eggs inoculated with theAGMK cell suspension. Since none of the harvest fluids exhibited anyhemagglutination when tested against guinea pig, chick or human RBC,this aspect of the embryonated egg study was considered satisfactory.

b. Yolk Sac Inoculation: Each of ten 6-day old embryonated eggs wasinoculated into the yolk sac with 0.5 ml of the AGMK cell suspension Lot#4742. Five (5) eggs were similarly inoculated with the original culturemedium and 5 eggs were included as uninoculated controls. Eggs wereincubated at 36° C. [±1° C.] for 9 days with periodic candling fordeaths. Of the medium-inoculated eggs, 2 were found dead (1 each on days2 & 5); and, of the AGMK-inoculated eggs, 2 were inadvertently broken.No other deaths or losses were recorded. Each group of yolk sacs (AGMKgroup=8; medium group=3; controls=5) was harvested, pooled, washed inPBS, emulsified by grinding and clarified by centrifugation at 2500 rpmfor 20 minutes at 20° C.

The 3 clarified suspensions were subpassaged via the same route intofresh 6-day old embryonated eggs as follows: AGMK group into 10 eggs;medium group into 5 eggs; control group into 5 eggs. An additional 5eggs were included as uninoculated controls. All eggs were incubated at36° C.±1° C. for 9 days with periodic candling for deaths. On day 7, 3eggs were found dead; 2 from the AGMK group and 1 from the medium group.There were no other deaths recorded.

This aspect of the embryonated egg study was considered satisfactory,because there was a greater than 80% overall survival (16 of 18) ofembryonated eggs (primary and subpassage) inoculated with the AGMK cellsuspension and viability of the egg was confirmed.

EXAMPLE 7 Use of the Characterized AGMK Cell Substrate for: (1)Isolation and Growth of Human Rotaviruses: and (2) Production of LiveVaccines Containing Such Viruses

The AGMK cells are the only characterized cells tested to date that aresuitable for vaccine purposes and support the efficient growth ofcompletely homologous human rotaviruses, i.e., rotaviruses that deriveeach of their 11 gene segments from a human rotavirus. This isparticularly important because a number of candidate homologous humanrotavirus vaccine strains are now under development, including thoseshown in Table V.

As seen in Table V, two candidate human vaccine rotaviruses of Gserotype 1 or G serotype 3 recovered from newborn infants were observedto grow to moderately high or high titer in AGMK cells, whereas, each ofthese neonatal rotavirus strains was markedly restricted in its capacityto grow in two types of cells certified for vaccine production, i.e.,FRhL₂ or Vero cells. In addition, one of these neonatal rotavirusstrains was also markedly restricted in replicative capacity in humanfetal fibroblast cell strain MRC-5 which is licensed for vaccineproduction.

Similar observations were made with two cold-adapted (ca) mutants ofhuman rotavirus of G serotype 1 or G serotype 2 that are also promisingcandidate live virus vaccine strains. Both human rotavirus mutants grewefficiently ire AGMK cells but were significantly or markedly restrictedin their growth in certified cells FRhL₂ and Vero.

A. Isolation and Serial Passage of Neonatal Human Rotavirus Strains

Initially, the toxicity of each new preparation of purified trypsin isdetermined for the AGMK cells to be tested. A known positive stoolextract is pre-treated with antibiotics (200 μg/ml gentamicin; 100 μg/mlneomycin; and 5 μg/ml fungizone) for 60 minutes at room temperature.Equal volumes of stool extract and trypsin at pre-determinedconcentration, e.g., 10 μg/ml, are mixed and allowed to incubate at 37°C. in a water bath for 30 minutes. In the interim, the cultures to beinoculated are washed three times with appropriate volumes (e.g., 3 mlfor roller tubes, 10 ml for T25 flasks, 30 ml for T75 flasks and 100 mlT175 flasks) of serum-free medium to dilute the serum that had been inthe growth medium in order to prevent components of serum frominactivating trypsin activity.

Roller tube cultures are inoculated with a 0.5 ml volume of thestool/trypsin mixture which is allowed to adsorb at 36° C.±1° C. for 60minutes while rotating on a roller drum apparatus. Cultures are washedonce and fed with 3 ml volumes of (EMEM) containing 1% of 10× SPG(sucrose, 2.18M; KH₂ PO₄, 0.038M; K₂ HPO₄, 0.072M; and mono-sodiumglutamate, 0.054M) and 1 μg/ml trypsin. Cell cultures in which trypsinis omitted are also included to control for the effect of trypsin.Incubation is at 36° C.±1° C.

Cultures are examined microscopically on a daily basis and cultures areharvested when cytopathic effect (CPE) that involves 75 to 100% of thecells is evident. Virus and control cultures are treated similarly with10% of 10× SPG (v/v) and subjected to one freeze-thaw cycle prior toharvest, sterility testing, distribution for storage, and serialpassage. All subsequent passages are carried out either in roller tubecultures or in 25 cm² flask cultures employing similar procedures butwith variation in trypsin concentration in both pre-treatment and in thefinal overlay.

An efficient system for producing virus plaques in AGMK cells isdeveloped using methods that are routine in the art so that the viruscan be plaque purified in order to obtain a homogeneous population ofvirus for subsequent vaccine development. The harvest fluids areroutinely titrated in the simian MA-104 cells to measure the virus yieldobtained. The virus is then triply-plaque-purified AGMK cells and theresulting clone is amplified by serial passage in progressively largertissue culture vessels. The conditions for maximum yield based ontrypsin concentration employed in both pretreatment and final overlay isdetermined for the progeny of the isolated clone using methods wellknown in the art.

B. Adaptation and Serial Passage Human Neonatal Rotaviruses or OtherHuman Rotavirus Strains

The human rotaviruses exhibit a very narrow tissue culture host range.As a consequence, isolation and serial passage of various strains areusually performed in commercially available laboratory cell culturessuch as simian MA-104 or primary monkey kidney, both of which areunsuitable for vaccine production. The former cells have not beenvalidated as a substrate for vaccine production and the latter cells arenotorious for their contamination with various simian microbial agentsincluding retroviruses. In contrast to MA104 or primary monkey kidneycells, cell substrates certified for vaccine production, such as simianFRhL-2 or Vero cells, either fail to support growth of completelyhomologous human rotaviruses or allow only poor growth.

Many well characterized rotaviruses have been isolated, seriallypassaged and triply plaque-purified in commercially available laboratorycell culture systems not suitable for vaccine production. In thoseinstances where it was necessary to use such viruses in vaccinedevelopment, the viruses were subsequently passaged and biologicallycloned in a cell substrate certified for vaccine development andproduction. Ideally, virus to be used in vaccines should be isolated andpassaged only in tissue culture cells certified to be acceptable forvaccine production. The AGMK cells of this invention meet thisrequirement because they support efficient growth of fully homologoushuman rotaviruses (Table V).

To develop a vaccine, routine studies are performed in flask cultures todetermine the conditions for maximum yield including trypsinconcentration required in both pre-treatment and final overlay. Onceadaptation of the vaccine virus to the AGMK cell substrate has beenachieved, the virus harvest is treated with ether (1 part ether to 4parts virus suspension for 60 minutes at room temperature followed by a30 minute bubbling with N₂ to drive off ether) to eliminate anyether-sensitive virus(es) that may have been present in the originalclinical specimen or acquired subsequently during passage in cellculture prior to initiation of vaccine development.

Live Virus Vaccine and/or Suspension Production

To produce vaccine, it is preferable also to prepare several backuppools of virus as follows:

1. Primary Seed Virus Pool

A pool of virus and serially passaged tissue culture control fluidssupplemented with 10% of 10× SPG (v/v) are prepared and, after sterilityis confirmed, and potency is determined, the fluids are distributed intosmall aliquots for storage at or below -70° C. to serve as 3rd levelback-up to final vaccine production. Total volume may range from 30 mlto 100 ml.

2. Secondary Seed Virus Pool

A pool of virus and serially passaged tissue culture control fluidssupplemented with 10% of 10× SPG (v/v) is prepared. After sterility isconfirmed, and potency determined, the fluids are distributed into smallaliquots for storage at or below -70° C. to serve as 2nd level back-upto final vaccine production. Total volume may range from 100 ml to 300ml.

3. Master Seed Virus Pool or Pre-Production Seed Virus Pool

A pool of virus and serially passaged tissue culture control fluidssupplemented with 10% of 10× SPG (v/v) is prepared. After sterility isconfirmed, potency is determined, and identity is confirmed by bothserology and electrophoretic pattern, the fluids are distributed intomulti-sized aliquots with storage at or below -70° C. These pools serveas first level back-up to final production. These fluids are subjectedto the Tissue Culture Purity (Safety) Testing simultaneously with thevaccine lot. Total volume may range from 500 ml to 1 liter.

4. Live Virus Vaccine Production

Volume to be produced is based on the number of doses and containersrequired plus a minimum of 400 ml of crude fluid for safety testing. Inaddition to the virus pool, the production of a small pool (200-400 ml)of the passaged tissue culture control fluid is recommended forsubsequent safety testing together with the crude virus harvest. Culturevessel fluids supplemented with 10% of 10× SPG (v/v) are subjected toone (1) freeze-thaw cycle prior to individual harvest and sterilitytesting. A sample pool is prepared and assayed for potency. Identity isconfirmed by both serology and electrophoretic pattern. The individualharvests are stored at or below -70° C. When sterility, potency andidentity are confirmed, crude harvests are thawed and pooled. Samples ofabout 400 ml are removed for safety testing. The remainder of the fluidis clarified by centrifugation at 1200 g for 20 min. at 5° C.,re-pooled, and distributed into final containers.

EXAMPLE 8 Adaptation of Human x Animal Reassortant Rotaviruses To Growthand Serial Passage in the Characterized AGMK Cell Substrate and the Useof Such Cells for Production of Live Virus Vaccines

Live attenuated rotavirus vaccines can be made using reassortantrotaviruses that derive ten of their eleven gene segments from theiranimal rotavirus parent and only one gene segment from their humanrotavirus parent (usually the gene that encodes the major protectiveantigen VP7). In one instance, human x rhesus monkey rotavirusreassortants are in a late stage of vaccine development and anapplication for a license has been submitted to the FDA.

All vaccine candidate human X animal reassortant rotaviruses studied todate have exhibited a very broad tissue culture host range attributableto the animal rotavirus parent, which contributes ten of its eleven genesegments to the reassortant. Although these vaccine candidatereassortants can proliferate to high titer in other cell lines certifiedfor vaccine production, comparative studies with the AGMK cell substrateindicate that the AGMK cell substrate provided a significant advantagein viral growth yield over FRhL2 cells in every instance tested (TableV). In addition, the AGMK cell substrate has proved to be superior toVero cells for the growth of human x rhesus rotavirus reassortants aswell as rhesus rotavirus itself (Table V). In these instances, RRV andits human rotavirus reassortants grew in the AGMK cell line to a titerthat was 16- to 100-fold higher than in FRhL2 cells and 10- to 1200-foldhigher than in Vero cells (Table V). The only instances in which theAGMK cell substrate was not superior to Vero cells involved the human xbovine rotavirus reassortants where viral growth yield in the two celllines was equivalent (Table V).

In addition to yield, the AGMK cell substrate has other advantages: (a)pre-treatment of virus with trypsin is not required; (b) theconcentration of trypsin required in culture medium is reduced; (c)there is a significant reduction in the amount of virus required in theinoculum; (d) incubation time is shortened; and (e) the infected cellmonolayer is completely lysed. Vaccine production is carried out asoutlined above.

EXAMPLE 9 Adaptation of Human Astrovirus to Growth, Serial Passage andSubsequent Live Virus Vaccine Production in the Characterized AGMK CellSubstrate

Previously, all human astroviruses have been isolated or propagated incells inappropriate or unacceptable for vaccine production. A Type 2strain of human astrovirus was successfully adapted to growth and serialpassage in the AGMK cell substrate from seed virus grown in the simianLLCMK₂, cell line. Flask cultures of AGMK cell grown virus were studiedto determine the optimal conditions for maximum yield based on trypsinconcentrations employed in both pre-treatment and final overlay. Onceadaptation was achieved, the virus harvest was treated with ether (1part ether to 4 parts virus suspension for 60 minutes at roomtemperature followed by a 30 minute bubbling with N₂ to drive off ether)to eliminate any ether sensitive simian virus that may have been pickedup during passage and plaquing in the commercially available laboratorycell culture systems. Fluid production was similar to that outlined forthe human rotavirus.

EXAMPLE 10 Propagation of Live Attenuated Poliovirus Vaccine Strains inthe Characterized AGMK Cell Substrate

The Sabin live attenuated oral poliovirus vaccine strains were grown inthe AGMK cells at passages 15 and 16. Each of the vaccine strains grewto high titer in AGMK cells (type 1 virus 10⁸.3 TCID₅₀ /ml, type 2 virus10⁸.1 TCID₅₀ /ml, and type 3 virus 10⁸.6 TCID₅₀ /ml). These titerstranslate into 100 to 1000 vaccine doses per ml of. tissue cultureharvest and are equivalent to the amount of virus produced by licensedprimary AGMK cells and MRC-5 cells.

EXAMPLE 11 Isolation and/or Adaptation of Enteroviruses to Growth,Serial Passage and Subsequent Live Virus Suspension and/or VaccineProduction in the Characterized AGMK Cell Substrate

Enteroviruses such as Coxsackie A9 virus were adapted to growth andserial passage in the characterized AGMK cells. These cells are used toproduce live virus suspensions and vaccine. Several seed virus poolshave been produced.

Live Virus Vaccine and/or Suspension Production

1. Primary Seed Virus Pool

A pool of virus and serially passaged tissue culture control fluidssupplemented with 10% of 10× SPG (v/v) are prepared and after sterilityis confirmed and potency is determined, the fluids are distributed intosmall aliquots for storage at or below -70° C. to serve as 3rd levelback-up to final vaccine production. Total volume may range from 30 mlto 100 ml.

2. Secondary Seed Virus Pool

A pool of virus and serially passaged tissue culture control fluidssupplemented with 10% of 10× SPG (v/v) are prepared. After sterility isconfirmed, and potency determined, the fluids are distributed into smallaliquots for storage at or below -70° C. to serve as 2nd level back-upto final vaccine production. Total volume may range from 100 ml to 300ml.

3. Master Seed Virus Pool or Pre-Production Seed Virus Pool

A pool of virus and serially passaged tissue culture control fluidssupplemented with 10% of 10× SPG (v/v) are prepared. After sterility isconfirmed, potency is determined, and identity confirmed by serology.The fluids are distributed into multi-sized aliquots with storage at orbelow ×70° C. These pools serve as first level back-up to finalproduction. These fluids are subjected to the Tissue Culture Purity(Safety) Testing simultaneously with the vaccine lot. Total volume mayrange from 500 ml to 1 liter.

4. Live Virus Vaccine Production

Volume to be produced is based on the number of doses and containersneeded plus a minimum of 400 ml of crude fluid for the required safetytesting. In addition to the virus pool, the production of a small pool(200-400 ml) of the passaged tissue culture control fluid is recommendedfor subsequent safety testing together with the crude virus harvest.Culture vessel fluids supplemented with 10% of 10× SPG (v/v) aresubjected to one (1) freeze-thaw cycle prior to individual harvest andsterility testing. A sample pool is prepared and assayed for potency andidentity is confirmed by serology. The individual harvests are stored ator below -70° C. When sterility, potency and identity are confirmed,crude harvests are thawed and pooled. Samples of about 400 ml areremoved for safety testing. The remainder of the fluid is clarified bycentrifugation at 1200 g for 20 min. at 5° C., re-pooled. anddistributed into final containers.

EXAMPLE 12 Isolation and Adaptation of Respiratory Syncytial Virus (ESV)to Growth and Serial Propagation in the Characterized AGMK CellSubstrate and Use of These Cells for Production of a Live Virus Vaccine

Respiratory syncytial viruses, subgroup A and B types, have been readilyisolated in these AGMK cell cultures directly from human nasal and/orthroat secretions. Specimens are centrifuged at 3000 rpm for 10 minutesto remove any particulate matter and then treated with antibiotics (200μg/ml gentamicin; 100 μg/ml neomycin; and 5 μg/ml fungizone) for 60minutes at room temperature. The antibiotic-treated specimens areinoculated into roller tube cultures of the AGMK cells and incubated at36° C.±1° C. on a roller drum. Once cytopathic effects are evident,cultures are harvested and further passages are carried out either inroller tubes or in flask cultures with subsequent serial passage andplaque purification. Subsequently, virus mutants are produced bychemical mutagenesis or are selected by passage at suboptimaltemperature using the AGMK cells in either instance. Live virus vaccineis then prepared using the AGMK cells.

Final viral yield of candidate live attenuated RSV vaccine strains grownin the AGMK cells was greater than that of these viruses grown in Verocells, which were previously the most efficient cell substrate certifiedfor production of RSV vaccines. An example is seen in Table VI, wherethe virus yield of a cold-passaged, temperature sensitive mutant of RSV(designated RSV A2 cpts248/404) is shown to be 4- to 16-fold higher inthe AGMK cell line than in Vero cells. Also, the amount of RSV producedby the human fetal diploid cell line MRC-5, which has been licensed foruse in production of virus vaccines, is 10- to 100-fold less than inAGMK cells. This mutant is highly attenuated but still capable ofinducing resistance to RSV in susceptible chimpanzees, which representthe most relevant animal surrogate for evaluation of attenuation andprotective efficacy of RSV mutants prior to initiation of clinicaltrials in humans.

Live attenuated RSV vaccines are designed to be used in very younginfants, the population at greatest risk of life-threatening RSVdisease. Because of the need to immunize such young individuals, thelive vaccine must be very attenuated (i.e., demonstrably weakened).Thus, it is noteworthy that the RSV A2 cpts248/404 mutant was able toattain a high titer in AGMK cells. Clearly, success of a vaccinecontaining this mutant and live RSV vaccines in general will requireattainment of a high level of viral yield.

To produce live virus vaccine, conditions for good growth and viralyield, such as temperature of incubation and inoculum size, aredetermined by routine methods with production proceeding as follows:

Live Virus Vaccine and/or Suspension Production

1. Primary Seed Virus Pool

A pool of virus and serially passaged tissue culture control fluidssupplemented with 10% of 10× SPG (v/v) are prepared and after sterilityis confirmed and potency is determined, the fluids are distributed intosmall aliquots for storage at or below -70° C. to serve as 3rd levelback-up to final vaccine production. Total volume may range from 30 mlto 100 ml.

2. Secondary Seed Virus Pool

A pool of virus and serially passaged tissue culture control fluidssupplemented with 10% of 10× SPG (v/v) is prepared. After sterility isconfirmed, and potency determined, the fluids are distributed into smallaliquots for storage at or below -70° C. to serve as 2nd level back-upto final vaccine production. Total volume may range from 100 ml to 300ml.

3. Master Seed Virus Pool or Pre-Production Seed Virus Pool

A pool of virus and serially passaged tissue culture control fluidssupplemented with 10% of 10× SPG (v/v) are prepared. Sterility isconfirmed, potency is determined, and identity confirmed by serology.The fluids are distributed into multi-sized aliquots with storage at orbelow -70° C. These pools serve as first level back-up to finalproduction. These fluids are subjected to the Tissue Culture Purity(Safety) Testing simultaneously with the vaccine lot. Total volume mayrange from 500 ml to 1 liter.

4. Live Virus Vaccine Production

Volume to be produced is based on the number of doses and containersneeded plus a minimum of 400 ml of crude fluid for the required safetytesting. In addition to the virus pool, the production of a small pool(200-400 ml) of the passaged tissue culture control fluid is recommendedfor subsequent safety testing together with the crude virus harvest.Culture vessel fluids supplemented with 10% of 10× SPG (v/v) areharvested directly without undergoing a freeze--thaw cycle. A samplepool is prepared and assayed for potency and identity is confirmed byserology. The crude harvests are pooled and aliquots of about 400 ml areremoved for safety testing. The remainder of the fluid is clarified bycentrifugation at 1200 g for 20 min. at 5° C., re-pooled, anddistributed into final containers.

EXAMPLE 13 Isolation and Adaptation of Parainfluenza Viruses to Growthand Serial Propagation in the Characterized AGMK Cell Substrate and Useof These Cells for Production of a Live Virus Vaccine

Parainfluenza viruses types 1, 2 and 3 were isolated previously inprimary AGMK cell cultures directly from human nasal and/or throatspecimens. Subsequently, virus types 1, 2 and 3 were readily adapted togrowth in the characterized AGMK cells. For isolation in thecharacterized AGMK cell substrate clinical specimens are centrifuged at3000 rpm for 10 minutes to remove any particulate matter and thentreated with antibiotics (200 μg/ml gentamicin; 100 μg/ml neomycin; and5 μg/ml fuigizone) for 60 minutes at room temperature. Theantibiotic-treated specimens are inoculated into the AGMK cell substrateroller tube cultures and incubated at 36° C.±1° C. on a roller drum.Once cytopathic effects and/or hemadsorption (with 0.1% guinea pig RBCin PBS, pH 7.2) is evident, cultures are harvested with further passagescarried out either in roller tubes or in flask cultures with subsequentserial passage followed by plaque purification. Subsequently, virusmutants are produced by chemical mutagenesis or are selected by passageat suboptimal temperature, using the AGMK cell substrate in eitherinstance.

An attenuated cold-adapted (ca) mutant of parainfluenza virus type 3(designated PIV3 ca cold passage 45) was successfully grown to hightiter in the AGMK cell substrate. Growth yield of the mutant was similarto the yield obtained in Vero cells, a certified simian cell line thathas not yet been licensed for production of live virus vaccine for usein humans. However, as shown in Table VII, final viral yields in theAGMK cells were approximately 30-fold higher than in the commonly used,certified fetal rhesus lung cell strain, FRhL-2, which also has not beenlicensed as yet for production of human viral vaccines.

The PIV3 ca cold passage 45 mutant, which is now in Phase II clinicaltrials, is satisfactorily attenuated for young infants who have not beenpreviously infected with naturally occurring PIV3. Although the mutantis very attenuated in young infants, it nonetheless stimulatesproduction of protective levels of serum PIV3 antibodies. Based upon theresults of clinical trials of the ca mutant in young infants, wecalculate that 1 ml of the viral yield from infected AGMK cells contains30 vaccine doses of PIV3 ca cold passage 45. Attainment of this level ofviral growth during vaccine production is particularly noteworthybecause the live PIV3 vaccine virus has been weakened so that is can beadministered safely to infants and young children, the target populationfor immunization.

To produce live virus vaccine, conditions for good growth and viralyield, such as temperature of incubation and inoculum size, aredetermined by routine methods with production proceedings as follows:

Live Virus Vaccine and/or Suspension Production

1. Primary Seed Virus Pool

A pool of virus and serially passaged tissue culture control fluidssupplemented with 10% of 10× SPG (v/v) are prepared and, after sterilityis confirmed and potency is determined, the fluids are distributed intosmall aliquots for storage at or below -70° C. to serve as 3rd levelback-up to final vaccine production. Total volume may range from 30 mlto 100 ml.

2. Secondary Seed Virus Pool

A pool of virus and serially passaged tissue culture control fluidssupplemented with 10% of 10× SPG (v/v) is prepared. After sterility isconfirmed, and potency determined, the fluids are distributed into smallaliquots for storage at or below -70° C. to serve as 2nd level back-upto final vaccine production. Total volume may range from 100 ml to 300ml.

3. Master Seed Virus Pool or Pre-Production Seed Virus Pool

A pool of virus and serially passaged tissue culture control fluidssupplemented with 10% of 10× SPG (v/v) are prepared. Sterility isconfirmed, potency is determined, and identity confirmed by serology.The fluids are distributed into multi-sized aliquots with storage at orbelow -70° C. These pools serve as first level back-up to finalproduction. These fluids are subjected to the Tissue Culture Purity(Safety) Testing simultaneously with the vaccine lot. Total volume mayrange from 500 ml to 1 liter.

4. Live Virus Vaccine Production

Volume to be produced is based on the number of doses and containersneeded plus a minimum of 400 ml of crude fluid for the required safetytesting. In addition to the virus pool, the production of a small pool(200-400 ml) of the passaged tissue culture control fluid is recommendedfor subsequent safety testing together with the crude virus harvest.Culture vessel fluids supplemented with 10% of 10× SPG (v/v) aresubjected to one (1) freeze-thaw cycle prior to individual harvest andsterility testing. A sample pool is prepared and assayed for potency andidentity. The individual harvests are stored at or below -70° C. Whensterility, potency and identity are confirmed, crude harvests are thawedand pooled. Samples of about 400 ml are removed for safety testing. Theremainder of the fluid is clarified by centrifugation at 1200 g for 20min. at 5° C., re-pooled, and distributed into final containers.

EXAMPLE 14 Isolation and/or Adaptation of Influenza Viruses to Growth inthe Characterized AGMK Cell Substrate and Use of These Cells forProduction of a Live or Inactivated Virus Vaccine

Influenza viruses type A, subtypes H3N2, and H1N1 and type B, have beenreadily adapted to growth in this characterized AGMK cell line. Asignificant advantage of isolating and growing influenza viruses in amammalian cell line rather than in embryonated chicken eggs stems fromthe fact that the hemagglutinin (the major protective viral protein ofinfluenza virus) retains the same amino acid sequence and conformationas that present in a virus isolated from humans. In contrast, mutantsbearing mutations affecting the hemagglutinin are selected duringadaptation of virus to growth in eggs. These mutations confer upon themutant a growth advantage in eggs.

The advantages of producing influenza virus vaccines, either live orkilled, in a mammalian tissue culture system rather than in embryonatedeggs derive from retention of the major protective antigenic sites onthe influenza virus hemagglutinin (HA) when virus is grown in mammaliamtissue culture. Adaptation of influenza A or B virus to efficient growthin embryonated chicken eggs frequently results in selection of viralmutants that possess mutations affecting one of the major protectiveantigenic sites on the viral HA, Loss of such an HA epitope that inducesprotective antibodies diminishes the effectiveness of a vaccinecontaining this form of viral antigenic mutant. A number of studies haveshown that influenza viruses isolated and propagated only in mammaliancell culture are more closely related antigenically to naturallycirculating viruses than are egg-adapted influenza virus strains. Forthis reason it would be advantageous to isolate and maintain influenza Aand B virus vaccine strains in a mammalian cell culture system,preferably primate, so as to maintain authentic antigenic structure ofHA which is the major protective protein of these viruses.

As shown in Table VIII, a variety of influenza A viruses of subtype H1N1or H3N2 grew efficiently in the AGMK cell substrate. Various influenza Bvirus strains also grow in the AGMK cells, albeit somewhat less wellthan the influenza A viruses. Since egg-propagated viruses were used asthe inoculum in this experiment, the viral yields shown in Table VIIIare minimal estimates because egg adaptation would be expected to selectfor mutants able to grow efficiently in eggs. Such mutants often growless well in primate cells than the original naturally occurring virusas a consequence of the mutations responsible for egg adaptation.

The AGMK cells can be used to isolate influenza A and B viruses frominfected humans and serve as a cell substrate for production of virusthat is used to produce an inactivated vaccine. Alternatively, the AGMKcells can be used to isolate and propagate virus during the derivationof attenuated mutants that are used for formulation of a live attenuatedvirus vaccine.

EXAMPLE 15 Isolation and/or Adaptation of Human and Simian Hepatitis AViruses to Growth, Serial Passage and Subsequent Live and InactivatedVirus Vaccine Production in the Characterized AGMK Cell Substrate

Wild-type hepatitis A virus strains of human origin grow poorly or notat all in cell culture on primary isolation: they must be adapted togrow efficiently in cell culture by the laborious procedure of serialpassage. In contrast, wild-type HAV of simian origin replicatesmoderately well during primary isolation in certain primate cells.However, certified or licensed cell culture systems suitable for primaryisolation and adaptation of HAV strains of human or simian origin arenot readily available or do not support the replication of HAV to highlevels. Although the examples are for human HAV, it will be obvious toone skilled in the art that the techniques can be applied to HAV strainsof simian origin.

Human hepatitis A virus, strain HM-175 (HAV/7). a strain suitable for acandidate live attenuated vaccine as well as a candidate inactivatedvaccine, was compared for ability to replicate in AGMK cells, FRhK-4(clone 11-1, the most sensitive cell line for replicating this virus todate), and licensed MRC-5 human diploid cells. Conditions for cellculture were those commonly used for the replication of HAV in FRhK-4cells.

As seen in Table IX and FIG. 3, HAV replicated to the same titer and atthe same rate in the AGMK cell substrate, which is suitable for vaccinedevelopment and production, as in FRhK-4, clone 11-1 cells, which arenot suitable for vaccine development. Furthermore, the size of the HAVplaques, as measured by the radioimmunofocus assay, was the same in bothcell types. In contrast, HAV strain HM-175 (HAV/7) did not replicateefficiently in MRC5 cells (a cell strain licensable for vaccinedevelopment) without extensive adaptation by serial passage.

Live Virus Vaccine and/or Suspension Production

1. Primary Seed Virus Pool

A pool of virus and serially passaged tissue culture control fluidssupplemented with 10% of 10× SPG (v/v) are prepared and after sterilityis confirmed and potency is determined, the fluids are distributed intosmall aliquots for storage at or below -70° C. to serve as 3rd levelback-up to final vaccine production. Total volume may range from 30 mlto 100 ml.

2. Secondary Seed Virus Pool

A pool of virus and serially passaged tissue culture control fluidssupplemented with 10% of 10× SPG (v/v) are prepared. After sterility isconfirmed and potency determined, the fluids are distributed into smallaliquots for storage at or below -70° C. to serve as 2nd level back-upto final vaccine production. Total volume may range from 100 ml to 300ml.

3. Master Seed Virus Pool or Pre-Production Seed Virus Pool

A pool of virus and serially passaged tissue culture control fluidssupplemented with 10% of 10× SPG (v/v) is prepared. Sterility isconfirmed, potency is determined, and identity confirmed by serology.The fluids are distributed into multi-sized aliquots with storage at orbelow -70° C. These pools serve as first level back-up to finalproduction. These fluids are subjected to the Tissue Culture Purity(Safety) Testing simultaneously with the vaccine lot. Total volume mayrange from 500 ml to 1 liter.

4. Live Virus Vaccine Production

Volume to be produced is based on the number of doses and containersneeded plus a minimum of 400 ml of crude fluid for the required safetytesting. In addition to the virus pool, the production of a small pool(200-400 ml) of the passaged tissue culture control fluid is recommendedfor subsequent safety testing together with the crude virus harvest.Culture vessel fluids supplemented with 10% of 10× SPG (v/v) aresubjected to one (1) freeze-thaw cycle prior to individual harvest andsterility testing. A sample pool is prepared and assayed for potency andidentity. The individual harvests are stored at or below -70° C. Whensterility, potency and identity are confirmed, crude harvests are thawedand pooled. Samples of about 400 ml are removed for safety testing. Theremainder of the fluid is clarified by centrifugation at 1200 g for 20min. at 5° C., re-pooled, and distributed into final containers.

Inactivated Virus Vaccine and/or Suspension Production

1. Primary Seed Virus Pool

A pool of virus and serially passaged tissue culture control fluidssupplemented with 10% of 10× SPG [v/v] that are sterility confirmed,potency determined and distributed into small aliquots for storage at-70° C., or below, to serve as 3rd level back-up to final vaccineproduction. Total volume may range from 30 ml to 100 ml.

2. Secondary Seed Virus Pool

A pool of virus and serially passaged tissue culture control fluidssupplemented with 10% of 10× SPG [v/v] that are sterility confirmed,potency determined and distributed into small aliquots with storage at-70° C., or below, to serve as 2nd level back-up to final vaccineproduction. Total volume may range from 100 ml to 300 ml.

3. Master Seed Virus Pool or Pre-Production Seed Virus Pool

A pool of virus and serially passaged tissue culture control fluidssupplemented with 10% of 10× SPG [v/v] that are sterility confirmed,potency determined, identity confirmed, and distributed into multi-sizedaliquots with storage at -70° C., or below. Pools serve as 1st levelback-up to final production. Fluids to be subjected to the TissueCulture Purity (Safety) Testing simultaneously with the vaccine lot.Total volume may range from 500 ml to 1 liter.

4. Inactivated Virus Vaccine Production

Volume to be produced is based on the number of doses and containerscontracted for plus a minimum of 400 ml of crude fluid needed for therequired safety testing. In addition to the virus pool, the productionof a small pool [200-400 ml] of the passaged tissue culture controlfluid is recommended for subsequent safety testing together with thecrude virus harvest. Culture vessel fluids supplemented with 10% of 10×SPG [v/v] are subjected to one (1) freeze-thaw cycle prior to individualharvest and sterility testing. A sample pool is prepared and assayed forpotency and identity and the individual harvests stored at -70° C., orbelow. When sterility, potency and identity are confirmed, crudeharvests are thawed, pooled, clarified and purified before inactivationwith formalin under standard conditions. The vaccine is then distributedinto final containers and safety-tested for sterility.

The novel African Green Monkey Kidney cell line described, supra, wasdeposited with the American Type Culture Collection on Nov. 4, 1994, andaccorded ATCC Designation No. CRL 11756. This deposit was made inaccordance with the Budapest Treaty. This cell line will be irrevocably,and without restriction or condition, released to the public upon theissuance of a patent to this application.

                  TABLE V                                                         ______________________________________                                        Comparison of Growth Patterns of Candidate                                      Rotavirus Vaccines in Various Cell Cultures                                                  Titer of Indicated Candidate Live Vaccine                       Virus Strain Produced in Indicated Cell                                      Candidate Rotavirus Culture as Determined by                                  Vaccine Plaque Assay in MA104 Cells (log.sub.10 pfu/ml).sup.a               Strain (Serotype).sup.b                                                                    AGMK.sup.c                                                                            FRhL-2    Vero.sup.c                                                                            MRC5.sup.c                             ______________________________________                                        Human Strains                                                                   D - ca30° C. (G1;P1A).sup.d 6.1 No Growth 4.7 NT.sup.e                 DS-1 - ca30° C. 6.3 No Growth No Growth NT                             (G2;P1B).sup.d                                                                M37 - neonatal (G1;P2) 5.2 No Growth No Growth NT                             Neonatal (G3;P2) 5.8 Inconsistent, Inconsistent, No                             poor growth poor growth Growth                                              Human × Bovine                                                          Viral Reassortants.sup.f                                                      D × UK(G1;P7) 7.1 5.8 7.1 NT                                            DS-1 × UK (G2;P7) 7.3 5.3 7.0 NT                                        P × UK-2 (G3;P7) 7.0 5.4 6.9 NT                                         ST-3 × UK-2 (G4;P7) 7.2 5.8 6.6 NT                                      Rhesus Rotavirus or                                                           Human × Rhesus                                                          Monkey Viral Reassor-                                                         tants.sup.f                                                                   D × RRV (G1;P5B) 7.9.sup.g 6.7 6.6.sup.g NT                              7.9  5.7                                                                      8.3  5.1                                                                     DS-1 × RRV (G2;P5B) 7.6.sup.g 5.7 6.6.sup.g NT                           7.6  6.1                                                                      7.7  4.8                                                                     RRV-2 (G3;P5B) 8.5.sup.g 6.7 7.2.sup.g NT                                      8.6  7.3                                                                      8.7  6.4                                                                     ST3 × RRV (G4;P5B) 7.6.sup.g 6.1 6.3.sup.g NT                            7.8  5.8                                                                      7.7  4.6                                                                   ______________________________________                                         .sup.a Virus suspension grown in indicated cells.                             .sup.b Serotype denotes viral outer capsid proteins (G and P) that induce     protective neutralizing antibodies.                                           .sup.c AGMK = African green monkey kidney cells (derived from                 Cercopithecus ethiopus monkey); FRhL2 = Fetal rhesuslung cells; MRC5 =        fibroblastic cells derived from fetal human lung. FRhL2 and MRC5 are          semicontinuous cell strains and can not be used beyond passage 32 for         vaccine production; Vero is a continuous cell line; the number of times       AGMK cells can be passaged serially and remain suitable for use in vaccin     development has not been determined yet but is at  # least 16.                .sup.d Coldadapted (ca) mutants of human rotavirus selected by growth and     serial passage in cell culture at suboptimal temperature that does not        allow efficient growth of naturally occurring (i.e., wild type) human         rotavirus.                                                                    .sup.e NT = not tested.                                                       .sup.f A single gene (encoding G serotype) derived from human rotavirus       and all the remaining 10 genes derived from bovine or rhesus rotavirus.       .sup.g Growth yield from second passage in AGMK or Vero cells of virus        previously passaged in FRhL2 cells; values shown indicate growth yield        from cell culture inoculated with 1:10; 1:00 or 1:1000 dilution of first      passage material, respectively.                                               Note:                                                                         AGMK cells were used at passages 10, 11, 14 or 15.                       

                  TABLE VI                                                        ______________________________________                                        Efficient Replication of a Candidate Live Attenuated RSV Vaccine Strain        (RSV A2 cpts248/404) in AGMK Substrate                                            Vero Cell Grown RSV                                                                             Quantity of Virus                                        A2 cpts248/404 (Log.sub.10 pfu/ml) Produced in                                Used to Initiate Cell Cultures Inoculated With*                             Infection in       1 ml   5 ml                                                ______________________________________                                        Vero Cells         5.80   5.59                                                  AGMK Cells 6.43 6.80                                                        ______________________________________                                         *RSV A2 cpts248/404 mutant grown and titrated at 32° C. Titrations     performed in HEp2 cells at 32° C.                                      Note:                                                                         AGMK cells used at passages 14 and 15.                                   

                  TABLE VII                                                       ______________________________________                                        Efficient Replication of a Candidate Live Attenuated Parainfluenza Virus       Type 3 Vaccine Strain (PIV3 Cold-Adapted Cold Passage 45) in                  AGMK Cell Substrate                                                            Temperature  Cell Substrate                                                                           Quantity of                                                                             Hemagglutinin                               of Incubation Used for Virus Produced.sup.b Titer                             of PIV3 ca cp45 Virus Growth.sup.a (Log.sub.10 pfu/ml) (Reciprocal)         ______________________________________                                        32° C.                                                                            AGMK       7.37        256                                            Vero 7.05 256                                                                30° C. AGMK 7.49 256                                                    Vero 7.22 128                                                                26° C. AGMK 7.54 256                                                    Vero 7.78 512                                                              ______________________________________                                         .sup.a Inoculum was FRhL2 cell grown virus currently being evaluated in       clinical trials. The titer of this FRhL2 cell virus suspension was            10.sup.6.0 pfu/ml.                                                            .sup.b Inoculated cell cultures harvested after 14 days of incubation         except 32° C. AGMK cultures which were harvested at 7 days because     of accelerated development of viral cell destructive effects.                 Note:                                                                         AGMK cells used at passages 13 and 14.                                   

                  TABLE VIII                                                      ______________________________________                                        Growth of Human Influenza A and B Viruses in AGMK Cell Substrate                                            Quantity of Virus Pro-                              duced at the highest                                                         Number of passage level as De-                                                Passages in termined by Titration in                                         Type and AGMK Cells Indicated Cell Substrate                                Subtype                                                                              Virus Strain at 36° C..sup.a                                                                  AGMK    MDCK.sup.b                              ______________________________________                                        A H3N2 A/Wash/897/80                                                                              1         6.05    6.60                                       A/Korea/1/82 3 7.15 7.79                                                      A/Bethesda/1/85 2 6.48 6.90                                                   A/Los Angeles/2/87 3 7.16 7.76                                                A/Shandung/9/93 3 7.28 7.78                                                  A H1N1 A/Texas/1/85 1 5.31 4.87                                                A/Kawasaki/9/86 3 6.50 6.81                                                   A/Texas/36/91 1 5.23 6.04                                                    B B/Texas/1/84 1 <3.00 4.78                                                    B/Ann Arbor/1/86 1 3.78 5.58                                                  B/Victoria/2/87 1 <3.00 4.82                                                  B/Yamagata/16/86 1 4.41 5.72                                               ______________________________________                                         .sup.a Passage in AGMK cell substrate initiated with egg grown influenza      virus at a dilution of 10.sup.-3 or 10.sup.-4 . The inoculum for passage      in cell culture was also 10.sup.-3 or 10.sup.-4 (except B/Victoria/2/87       which was 10.sup.-2) in order to minimize generation of defective,            interfering virus particles. Tissue culture medium and agarose overlay in     plaque assays contained 0.5 μg/ml of trypsin.                              .sup.b MDCK is a continuous cell line of canine kidney cells often used       for titration of human influenza viruses.                                

                  TABLE IX                                                        ______________________________________                                        Ability of AGMK Cells to Support Efficient Growth and Plaque                    Formation of Candidate Hepatitis A Virus Vaccine Strain                       HM-175 (HAV/7)*                                                                          Cell Culture Used for Virus Replication                          HAV Property AGMK.sup.a                                                                              FRhK-4(11-1).sup.a                                                                       MRC-5.sup.a                                 ______________________________________                                        Titer (log.sub.10 pfu/ml)                                                                  7.4       7.4        Little or                                     of Virus Achieved in   No Growth.sup.c                                        Inoculated Culture.sup.b                                                      Plaque Size Large Large --                                                  ______________________________________                                         *Candidate strain for both inactivated and live attenuated HAV vaccines.      .sup.a AGMK = African green monkey kidney derived from Freeze #2129.          FRhK4(11-1) = HAVpermissive cell clone derived from fetal rhesus kidney4      cells (Funkhouser AW, Purcell RH, D'Hondt E, Emerson SU, Attenuated           hepatitis A virus: Genetic determinants of adaptation to growth in MRC5       cells. J Virol 68:148-157; 1994). MRC5 = fetal human lung fibroblast cell     substrate.                                                                    .sup.b Determined by plaque assay on AGMK and FRhKA(111) cells.               .sup.c Growth not detected by plaque assay performed with FRhK4(11-1) cel     culture.                                                                      Note:                                                                         AGMK cells used at passages 12, 13, 15 and 16.                           

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 4                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - CCTCAGAGCT GCATAAAAGC AGAT          - #                  - #                    24                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - TCACTCAAGT CCCTGTTCGG GCGC          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - TACTAGGAGA CCAGCTTGAG CCTG          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - TGCTGGAGTT TCTCTCGCCT GGGT          - #                  - #                    24                                                                    __________________________________________________________________________

What is claimed is the following:
 1. A method for producing a virus selected from the group consisting of rotaviruses, enteroviruses, polioviruses, respiratory viruses, astroviruses, and hepatitis A viruses, using a cell line originating from the kidney of an African Green Monkey, wherein said cell line supports the growth of rotaviruses, enteroviruses, polioviruses, respiratory viruses, astroviruses, and hepatitis A viruses, and wherein said cell line is substantially free of viable adventitious microbial agents, said method comprising the steps of:1. inoculating said cell line with a sample containing an inoculum of said virus;
 2. incubating said inoculated cell line to permit proliferation of said virus; and
 3. harvesting the virus resulting from step
 2. 2. A method, according to claim 1, wherein the virus is a human rotavirus.
 3. A method according to claim 1, wherein said virus is a human X animal rotavirus reassortant.
 4. A method, according to claim 1, wherein the virus is a human astrovirus.
 5. A method, according to claim 1, wherein the virus is an enterovirus.
 6. A method according to claim 1, wherein the virus is a Sabin live attenuated oral polio virus vaccine strain.
 7. A method, according to claim 1, wherein the virus is a respiratory virus.
 8. A method, according to claim 1, wherein the virus is a hepatitis A virus. 